Material and methods:

1. CAF01 adjuvant increases the protection conferred by a commercially available influenza split vaccine in a ferret model C.J.M. Martel a*, T.H. Jensen a, L.P. Nielsen b, E.M.Agger b, M. Blixenkrone-Møller a, P.Andersen b, B. Aasted a aDepartment of Veterinary Disease Biology, Faculty of Life Sciences, University of Copenhagen, Stigbøjlen 7, DK-1870 Frederiksberg C, Denmark. b Department of Infectious Disease Immunology, Statens Serum Institut, Copenhagen, Denmark. Abstract:The immunogenicity and protective efficacy of current preventive vaccines against influenza are considered suboptimal, and the development of novel effective influenza vaccination strategies is urgently needed. Commercially available trivalent split vaccines are known to elicit mainly a humoral immune response, whereas the induction of cell-mediated immune responses is negligible. Recently, a cationic liposomal adjuvant (CAF01, dimethyldioctadecylammonium/trehalose 6,6’-dibehenate) was developed. In the current study, we compared the immune response in ferrets vaccinated with a commercially available influenza split vaccine with the same vaccine mixed with the CAF01 adjuvant and furthermore used two recently circulating H1N1 viruses for the challenge of the animals. CAF01 improved the immunogenicity of the vaccine, increasing the influenza-specific IgA and IgG levels as well as triggering cellular-mediated immunity, measured by flow cytometry as the production of interferon-gamma by lymphocytes. The adjuvant also enhanced the protection conferred by the vaccine, reducing the viral load measured in nasal washes by RT-PCR. The protection data obtained in the human relevant challenge model supports the potential of CAF01 in future influenza vaccines. • Material and methods: • 24 ferrets were immunized twice at two-weeks intervals, then challenged after a month of rest with 107 TCID of an homologous H1N1 virus. • Vaxigrip: 8 ferrets, 2 x 80 µl commercial vaccine • Vaccine + adjuvant group: 8 ferrets, 2 x 80 µl vaccine + 250 µl CAF01 • Control PBS group: 8 ferrets, 2 x 250 µl PBS • Viral titers in nasal washes were measured by RT-PCR, Influenza-specific antibodies were detected in serum via ELISA using homemade rabbit anti-ferret IgG, PBL positive for intracellular gamma-IFN staining after PMA stimulation were analyzed by flow cytometry. • In a similar experiment, 24 ferrets received various doses of influenza vaccine with or without CAF01 and then were challenged with a homologous H1N1 virus. Results and Conclusions C. Figure 1. Immune response to vaccine antigens. A. Serum IgG antibodies measured by IgG specific ELISA B. Hemagglutination inhibition assay C.Percentage of IFNg positive lymphocytes at week after 24 hours of stimulation in cell culture with a recombinant H1 hemagglutinin Figure 2. Viral excretion A. Number of viral copies found in nasal washes of infected animals measured by quantitative RT-PCR. B. Number of copies in nasal washes of individual infected animals at peak replication day (day 3) C. Percentage of animals excreting the virus in their nasal washes during the challenge period (cumulative results of 3 similar experiments, for a total of 24 animals in each group). Figure 3. A/Brisbane/59/2007 study. A. Vaccine-specific serum IgG titers measured in serum by ELISA. B. Hemagglutination inhibition assay serum titers. C. Vaccine-specific IgAtiters measured in nasal washes by ELISA. D. Number of viral copies found in nasal washes of individual infected animals at peak replication days (day 3 and 4), measured by quantitative RT-PCR. CAF01 enhances antibody and CMI responses of the split vaccine significant increase of vaccine-specific IgG antibodies titers in serum (fig 1A and 1 B) significant increase in the percentage of IFN-g positive lymphocytes (fig 1C) CAF01-adjuvanted split vaccine confers protection against H1N1 influenza challenge median number of viral copies 10 to 100-fold lower than vaccine only and the mock-vaccinated (fig 2A) CAF01 allows for a reduction in vaccine dose 0.15 µgdose of vaccine adjuvanted with CAF01 reduced the viral load to the same level as observed with the unadjuvanted 15 µgdose. With the 15 µgdose,the adjuvant led to a 100-fold reduction of the viral load compared to the same dose of vaccine without CAF01 (fig 3 D).